NGS-based microbial contaminant identification
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During the last decades whole genome and metagenome analysis have provided a wealth of knowledge. However, these studies depend heavily on high-molecular weight, sufficient concentration, adequate quality and unbiased DNA extration methods. As different DNA extraction methods differ in their principle of obtaining nucleic acids and the outcome in terms of quantity as well as quality, this blog describes basic considerations that can help you select an appropriate extraction method for your sequencing project. Moreover, here we have a special focus on extractions for meta-omics related approaches to study complex microbial communities, such as those from faeces, soil or food fermentation processes.
Obtaining sufficient amounts of nucleic acids with high quality are basic criteria to evaluate the performance of a extraction methods. Nucleic acid amounts are easily measured by photometric assays or, even more accurately, using fluorometric assays that are less hampered by compounds that are carried over from the original sample (e.g. carbohydrates) or introduced during the extraction (e.g. phenol) and influence photometric measurements. Furthermore, conventional agarose gel electrophoresis or more modern bio analyser devices provide the means to evaluate the size distribution of extracted nucleic acids. High molecular weight nucleic acids are preferred or even required for library preparation of single genomes or metagenomes and subsequent sequencing on the Illumina and PacBio sequencing platforms.
While these criteria are the essential for any DNA based techniques, (16S ribosomal rRNA (rRNA) targeted) microbial profiling and meta–omics related approaches additionally require that the nucleic acids represent the microbial community in the environmental samples (e.g. water, soil, faeces, and food) from which the nucleic acids were extracted. However, the latter is hampered by the irregular biostructure of environmental samples that contains an unevenly mixed microbial communities (Swidsinski, et al., 2008; Salonen, et al., 2010).
Existing extraction methods and commercially available kits differ in their degree of homogenization, mode of cell lysis (enzymatic and/or mechanical), and nucleic acid recovery principle (e.g. column based or by precipitation; Quigley, et al., 2012). Although, mechanical lysis by bead beating to homogenize samples offers a better penetration of the lysis buffer through the complete sample, it may result in a higher degree of DNA shearing. The latter may be detrimental to whole genome sequencing endeavours that rely on input of high molecular weight DNA (Salonen, et al., 2010; Henderson, et al., 2013). However, methods employing a bead beating step do appear to generate largest DNA yields with higher microbial (Bacteria, Archaea, and fungi) numbers and diversity estimates (Salonen, et al., 2010; Henderson, et al., 2013, Quigley, et al., 2012). Shearing of the DNA can be minimized by removing the aqueous phase after each round of beat beating that are employed to lyse remaining cells. (Salonen, et al., 2010; Yu and Morrison, 2004). Though chemical and enzymatic lysis methods are gentler, they do not fully access all microbial populations in the sample owing to the irregular biostructure described above.
Our own evaluation of different bead beat apparatuses revealed that the apparatus itself as well as homogenization time indeed has a profound influence on the overall DNA yield (Figure 1). However, extracted DNA amounts did not show an gradual increase with more and longer homogenization steps, which is most likely due to the fragment specifics used for extraction. Analysis of the DNA by agarose electrophoresis showed that even at high speeds and prolonged homogenization, most DNA was high molecular weight (Figure 2).
Figure 1. Extracted DNA yield with differing homogenization time and apparatus employed for DNA extraction from a single faecal sample (A) and agarose gel with 5 µl extracted DNA after 30 minutes of electrophoresis (B). Apparatus 1 was used at its maximum speed of 30 vibrations per second, while apparatus 2 was used at one of its intermediate speeds of 5500 rpm.
Furthermore, we tested commercially available DNA extraction kits on several different faecal samples and measured DNA yield using a fluorometric essay. This revealed that there were profound differences in terms of DNA yield (Figure 2), indicating that the choice of DNA extraction method influences how much starting material is required and practical progress of the sequencing project in terms of speed.
Figure 2. Comparison of two commercially available kits for DNA extraction of DNA from faeces collected from human adult (A) and human baby (B). Samples were stored at room temperature (RT), frozen at -80°C, and in a DNA stabilization buffer (SB) prior to DNA extraction.
It is far from trivial to assess which extraction method yields the nucleic acid fraction that represents the true microbial composition most accurately (Henderson, et al., 2013). Although previous studies have shown that the extraction methodology itself can have a profound impact on the outcome of microbial profiling studies, this impact is only minor compared to the variation between, for example, faecal samples from different individuals (Salonen, et al., 2010, Henderson, et al., 2013). Nonetheless, the method of extraction can have a strong bias on the quantification of total bacteria and abundance estimations of specific taxonomic groups (e.g. Actinobacteria and Bacteroidetes; Salonen, et al., 2010, Henderson, et al., 2013). These observations affirm that the extraction methodology is a strong determinant for the outcome of microbial profiling and other DNA/RNA based techniques, including metagenomics and -transcriptomics. One approach to evaluate the efficiency of the nucleic acid extraction method is to determine the composition of micro-organisms in samples of interest through sequencing of 16S rRNA or its encoding gene. Though it would be impossible determining whether the obtained microbial composition represents the true microbial community, this data would allow for calculation of richness and diversity estimates (See Lozupone and Knight, 2008 for a review). In theory, higher estimates would indicate that nucleic acids were extracted from more different organisms compared to relatively lower estimates for samples. The latter would indicate that extraction was not efficient for all organisms present in the sample.
In conclusion, to evaluate the applicability of nucleic acid extraction one should not only consider quantity and quality as essential criteria, but also an optimal microbial diversity. The latter is of importance when assessing the microbial composition and function/activity through microbial profiling and meta-omics approaches, respectively. BaseClear provides solutions to extract DNA as well as RNA from complex communities, such as those from faecal samples and can collaborate to set up extraction methods yielding material that would be suitable for library preparation and sequencing.
Tom van den Bogert (Ph.D.) – Product Specialist Metagenomics